Clinical Impact and Therapeutic Opportunity of Insulin Receptor Substrates 1/2 in Acute Myeloid Leukemia

Background: The identification of biological and clinical prognostic factors in acute myeloid leukemia (AML) allowed the definition of patient subgroups and the realization of risk-adapted and targeted treatment strategies. Insulin-like growth factor 1 receptor/Insulin Receptor Substrates (IGF1R/IRS) pathway plays an important role in the development of neoplasia. IRS1/2 activates AKT/mTOR and MAPK pathways, through their interaction with PI3K and GRB2, culminating in increasing cell proliferation. NT157 is an allosteric inhibitor of IGF1R-IRS1/2 signaling that showed antineoplastic effects in preclinical studies of solid tumors. However, IRS1/2 clinical function and NT157 effects were not assessed in AML. Aims: To investigate IRS1 and IRS2 mRNA expression in AML patients and their impact in clinical outcomes, and to analyze the effects of the NT157 in AML cell lines. Material and methods: Comparison of IRS1 (probe nº 204686) and IRS2 (probe nº209184_s) expression from 581 AML patients and 8 CD34+ cells from healthy subjects were analyzed using data from Amazonia! Platform. For survival analysis, IRS1 and IRS2 mRNA expression levels from 173 AML patients (92 male - median age 58 years [range: 18-65]) were obtained from TCGA AML study available online on CBioPortal for Cancer Genomics. NB4, NB4-R2, Kasumi-1 and THP1 cell lines were submitted to NT157 (0.5, 1.0, 2.0, 4.0, 8.0 or 16 µM) 72 hours and evaluated for cell viability (MTT assay), apoptosis (Annexin V/PI), cell cycle (PI), ROS production (DCFDA), mitochondria staining (MitoTracker), and protein expression/activation (western blot). Bone marrow mononuclear cells (BMMC) were obtained from 4 AML patients at diagnosis and submitted to cytotoxic assays. Statistical analyzes were performed using ANOVA, Mann-Whitney or Kruskal-Wallis and Spearman correlation tests, as appropriate. For survival analysis, Kaplan-Meyer curves were compared with the log-rank test. Cox regression analysis was also applied. Results: IRS1 expression, but not IRS2, predicted outcomes. Reduced IRS1 expression showed poorer disease-free survival (DFS) (survival median time [MT]: 10.1 months [mos] vs. 28.4 mos, P<0.001; Hazard ratio [HR]: 0.51 [CI95:0.32 - 0.79]) and overall survival (OS) (MT: 14.5 mos vs. 27.4 mos, P=0.009; HR: 0.61 [CI95:0.42 - 0.88]). IRS1 expression independently predicted poorer DFS (HR: 0.59 [CI95: 0.36 - 0.79]; P= 0.03) using cytogenetic risk stratification, age and leukocytes as confounders. Of note, IRS1 level was positively correlated with proapoptotic CD27 (r=0.51; P<0.001) and with IL17RA (r=0.62; P<0.001) related to CD34 cell differentiation. IRS2 expression was upregulated in AML harboring t(15;17) (n=36; P<0.01) and inv(16) (n=37; P=0.01) in comparison to CD34+. In NB4, NB4-R2 and Kasumi-1 cells, NT157≥0.5µM reduced cell viability (P<0.05) and increased apoptosis (P<0.05). The mean percentage of annexin V+ cells for control, NT157 2.5, 5 and 10µM were 11, 47, 73 and 75% for NB4, 11, 41, 69 and 75% for NB4-R2 and 17, 45, 61 and 64% for Kasumi-1, respectively. In TP53-null cell line THP1, NT157 reduced cell viability at doses higher than 2µM (P<0.05) and induced apoptosis at 10µM (9.1 vs. 25%; P<0.05). NT157 induced ROS production in NB4 (fold-increase of mean fluorescence intensity [MFI]: 25.8 and 24.8), NB4-R2 (MFI: 26.7 and 31.4), Kasumi-1 (MFI: 5.8 and 6.6) and THP1 (MFI: 1.8 and 4.1) at 5 and 10µM (all P<0.05) and increased mitochondrial mass in NB4 (MFI: 3.9 and 3.7), NB4-R2 (MFI: 2.6 and 2.9), Kasumi-1 (MFI: 3.2 and 4.7) and THP-1 (MFI: 2.6 and 2.2) (all P<0.05). NT157 also modulated cell cycle progression, as evidenced by G2/M arrest in THP-1 and sub-G0/G1 in other cell lines (P<0.05). The IGF1R-IRS1/2 inhibitor NT157 reduced activation/expression of IGF1R (Tyr1135), IRS1/2 (Tyr612), AKT1/2/3 (Ser473), P70S6K (Thr421/Ser424), 4EBP1 (Thr70), ERK1/2 (Thr185/Tyr187) and induced DNA damage (increased γH2AX). NT157 did not induce autophagy, as demonstrated by non-degradation of p62 and lack of conversion of LC3BI into LC3BII in cell lines tested. NT157≥0.5 µM reduced cell viability and induced apoptosis in BMMC from 4 AML patients in a dose dependent manner (P<0.05). Conclusions: In AML, downregulation of IRS1 predicted dismal prognosis and the IGF1R-IRS1/2 inhibitor NT157 exerted an antineoplastic activity, downregulated PI3K/AKT and MAPK signaling. IRS1/2 arises as a promising therapeutic target for AML patients.